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Journal of the Chilean Chemical Society

versão On-line ISSN 0717-9707

J. Chil. Chem. Soc. v.51 n.1 Concepción mar. 2006

http://dx.doi.org/10.4067/S0717-97072006000100013 

J. Chil. Chem. Soc., 51, Nº 1 (2006)

LIPID PEROXIDATION RATES OF DPPC LIPOSOMES CONTAINING DIFFERENT AMOUNTS OF OXIDABLE LIPIDS SHOW OPPOSITE DEPENDENCE WITH THE TEMPERATURE.

MARÍA CUBILLOS1, EDUARDO LISSI 2 AND ELSA ABUIN2

1 Present address Facultad de Ciencias de la Salud. Universidad Diego Portales. Ejercito 141. Santiago-Chile.

2 Facultad de Química y Biología, Universidad de Santiago de Chile. Casilla 40 - Correo 33. Santiago - Chile

E-mail: eabuin@lauca.usach.cl, maria.cubillos@udp.cl


ABSTRACT

The effect of temperature on the oxidation rates of arachidonic acid and linoleic acid incorporated into dipalmitoylphosphatidylcholine liposomes have been investigated. The reaction was started by the photolysis of 2,2'-azobis (2-amidino propane) in order to minimize the dependence of the initiation rate with temperature. Both positive and negative activation energies were obtained for the rate of the oxidation, depending on the amount of oxidable lipid (20% or 50% mol:mol) and the temperature range considered. Changes in the rigidity of the liposomes elicited by temperature were assessed by measuring the generalized polarization of 6-dodecanoyl-2 (dimethylamino naphthalene), Laurdan. The high increase in rate observed at low temperatures employing lipid mixtures that render liposomes in the gel phase is attributed to a reduced rate constant of the termination step of the oxidation chain.

Keywords: lipid peroxidation; linoleic acid; arachidonic acid; dipalmitoyl- phosphatidylcholine


 

INTRODUCTION

The oxidation of unsaturated lipids is a matter of great interest, both from the biological and food preservation points of view [1-4]. In cell membranes the lipids are highly organized, and this organization can strongly modify the rate and kinetics of the lipid peroxidation process. Liposomes has been widely employed as controlled microreactors that mimic the properties of the more complex biological membranes. They have the advantage that their properties can be manipulated almost at will by changing their size, composition and/or temperature. The kinetics of the oxidation process can be similar [5,6] or different [7-10] to that observed in homogeneous solutions, depending of a variety of factors [7,8]. In spite of the importance of the process, there are few studies regarding how the temperature affects the rate of a lipid peroxidation process that takes place in a highly organized liposomes. This dependence is considered interesting since the change in temperature can produced notable changes in the properties (i.e. organization and microviscosity) of the liposomes whose effects upon the reaction rate of the process are difficult to predict. Cervato et al. [11] have studied the peroxidation of arachidonic acid in different liposomes below and above their phase transition temperature [11]. In all the systems considered, the rate of oxidation decreased with temperature, being higher in the temperature range below the phase transition temperature. This suggests that the rate of the process is higher in the more rigid environment, a result supported by the fact that the oxidation rate (measured by malondiadehyde formation) was dramatically slower in DPPE than in DPPC liposomes. However, a clear interpretation of the data was precluded by the lack of control of the initiation process, due to the interaction of added Fe(II) with hydroperoxides present as impurities in the lipid mixture. In order to test the generality of this apparently anomalous result in a more controlled system, we have studied the oxidation of arachidonic acid (AA) and linoleic acid (LA) in dipalmitoylphosphatidylcholine (DPPC) liposomes initiated by the photolysis of 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) over a wide temperature range. The results show that both positive and negative apparent activation energies can be obtained, depending upon the type and concentration of the unsaturated lipid.

MATERIALS AND METHODS

Materials

Dipalmitoylphosphatidylcholine, DPPC (Sigma > 99 %), linoleic acid, LA (Sigma), arachidonic acid, AA, (Sigma), Tempol (Aldrich), a-tocopherol (Sigma), 6-dodecanoyl-2 (dimethylamino naphthalene), Laurdan (Molecular Probes), 2,2´-azobis (2-amidinopropane) dihydrochloride, AAPH, (Wako Chemicals), and chloroform (Merck, p.a.) were used as received. All experiments were performed in 10 mM Tris-HCl buffer, pH = 7.4.

Liposome preparation

Liposomes were prepared by adding in a round-bottomed tube, known amounts of LA or AA and DPPC dissolved in chloroform. The solvent was removed by slow evaporation at 40 0C on a water bath under a stream of nitrogen to obtain a thin film. Then an appropriate volume of the buffer was added to obtain the desired working concentration of the lipids. The suspensions were subjected to five frozen-thaw cycles, transferred into a home made extruder, and extruded 10 times back and forth through polycarbonate filters (0.4 mm nominal pore size, Nucleopore, Corning Costar) at 400C. This procedure favors the formation of unilamelar liposomes. Some liposomes were prepared by sonication. The procedure was the same as described before until the suspension of the lipids in buffer was obtained. The suspension was then subjected to ultrasonic irradiation with a probe type sonicator (Sonics & Materials, Inc., model VCX 600; 75 watts) for 15 min under continuous irradiation under a stream of nitrogen. The samples were centrifuged to remove titanium particles prior to its use.

Kinetic studies

Oxidations were carried out in air equilibrated solutions in the presence of 0.25 mM AAPH as initiator. The reaction was started by the photolysis of AAPH using a medium pressure mercury lamp coupled to a 360 nm filter. This procedure was employed in order to minimize the dependence of the initiation rate with temperature. The rate of the process was followed by measuring the oxygen concentration as a function of time using a Biological Oxygen Monitor YSI Model 5300 (containing a thermostatized cell) coupled to a Perkin Elmer Model R50 recorder. The values obtained in the presence of liposomes were corrected by subtracting the oxygen consumption rate measured in blank experiments (in presence of DPPC liposomes). In some experiments lipid soluble free radical scavengers (Tempol or a-tocopherol) were added to estimate the rate of chain initiation from the observed induction times.

Laurdan generalized polarization measurements

Laurdan generalized polarization measurements were performed employing an Aminco Bowman Series 2 luminiscence spectrometer. Generalized polarization (GP) values were calculated from the emission intensities registered at 440 and 490 nm by GP (I440 - I490) / (I440 + I490) after excitation of the samples at 340 nm.

RESULTS AND DISCUSSION

The presence of polyunsaturated lipids in the liposomes increases nearly ten times the rate of oxygen uptake, suggesting the occurrence of relatively short chain processes. This is further supported by the almost total lack of oxygen consumption elicited by the presence 16 mM a-tocopherol or Tempol. Similar induction times were obtained with both antioxidants, being ca. 2.6 times shorter at 60 ºC than at 28 ºC. The rather small dependence with the temperature is compatible with the fact that the photolysis of AAPH is the main source of free radicals under the employed conditions. This small dependence of the initiation rate with temperature is further confirmed by experiments carried out in absence of polyunsaturated lipids. In fact, the rate of this process, that is determined by the rate of free radical production, increases nearly two times when the temperature increases from 28 0C to 60 ºC.

The rate of oxygen uptake was determined in liposomes containing 20 % (mol:mol) or 50 % (mol:mol) of LA or AA incorporated to DPPC liposomes. A summary of the oxidation rates measured under steady state conditions are shown in Figures 1 and 2. The data of these figures show striking differences, depending on the amount of oxidable lipid present in the liposomes. The liposomes containing 20 % of unsaturated lipid show a noticeable decrease in oxidation rate when the temperature increases. Similar results were obtained in sonicated (smaller) liposomes (data not shown). These results closely resembles those reported by Cervato et al.[11]. Furthermore, it is important to note that the decrease in oxidation rate with temperature takes place in spite of a moderate increase in the initiation rate, as assessed from the decrease in induction time and increase in the rate of oxygen uptake in absence of unsaturated lipids.


Figure 1. Dependence of the oxygen uptake rate with the temperature for DPPC liposomes containing 20 % (mol:mol) of LA ( ) or AA () . Liposomes prepared by extrusion. Oxidation carried out in air saturated solutions employing the photolysis of 0.25 mM AAPH as initiation process.

Table 1. Oxidation rates of polyunsaturated lipids and PG of Laurdan
in DPPC liposomes measured at 23 ºC.

In order to assess if the anomalous Arrhenius dependence observed at low percentages of unsaturated lipids is due to the concomitant decrease in the rigidity of the liposomes, we measure the temperature dependence of Laurdan generalized polarization, PG [12,13]. The results obtained are shown in Figure 3. Since in the temperature range considered the rigidity of the liposomes decreases (Fig. 3), the present results imply that this rigidity favors the oxidation process. This increase in rate with the rigidity of the liposomes can explain the apparently anomalous result that, at low temperatures, the rate of the process decreases when the percentage of oxidable lipid increases (Table 1).

If it is accepted that oxidation in liposomes takes place by a conventional free radical mediated chain mechanism, the results given in Fig. 1 and in Table 1 can be explained if the termination process is more affected by the rigidity of the media than the propagation process. In other words, in the more rigid liposomes the chain process is particularly long, due to a noticeable decrease in the rate constant of the termination step. On the other hand, the results obtained in liposomes containing 50 % of unsaturated lipids, show the opposite dependence with the temperature. In fact, the data of Fig. 2 show an increase in oxidation rate with temperature upon all the temperature range considered. This increase in rate is compatible with an apparent activation energy of ca. 7 kcal/mol. It is interesting to note that, in fluid systems, the rate of the process increases when the amount of oxidable lipid increases (compare Figs. 1 and 2) while in the more rigid phase the rate decreases when the amount of oxidable lipid increases (Table 1)

It is difficult to completely establish the reason of the opposite behavior of liposomes containing 20 or 50 % of unsaturated lipids. However, analysis of the data allow to conclude that this observed difference is not due only to differences in the rigidity of the liposomes. This is emphasized by a comparison of the results obtained employing 20 % AL and 50 % AA. The data of Table 1 show that both systems have similar PG values and similar oxidation rates at 23 ºC. Furthermore, the data of Fig. 3 show that the change in PG between 20 and 40 ºC is very similar for both liposomes. However, in spite of these similarities they present an opposite dependence with temperature in the 20 to 40 ºC range. In fact, while the rate of the liposomes containing 20 % LA decrease with temperature (Fig. 1), that of the liposomes containing 50 % AA show the opposite dependence (Fig. 2). GP absorption and emission spectra were determined in order to assess if the different behaviour is due to the fact that phase separation takes place in some of the systems considered. The spectra obtained did not show any evidence of domains formation [12,13].


Figure 2. Idem than Figure 1 but employing liposomes prepared
with equimolar amounts of DPPC and LA ( ( ) or AA ().


Figure 3. Dependence with temperature of the generalized polarization of Laurdan in extruded DPPC liposomes containing 20 % () or 50% ;() of LA acid and 50 % AA (O). GP obtained from the fluorescence intensities measured at 440 nm (I440) and 490 nm (I490) according to GP = (I440 - I490)/( I440 + I490) . Excitation at 340 nm.

In conclusion, the present results show that the effect of temperature upon the oxidation rate of lipids markedly depends upon the properties of the system and can drastically change with the proportion of oxidable lipid. In particular, in lipid mixtures of low polyunsaturated lipids content, the oxidation rate decreases when the temperature and/or the proportion of oxidable lipids increase. This anomalous behaviour can be explained, at least partially, by a drastic decrease in the termination step of the oxidation chain process in the more rigid liposomes.

ACKNOWLEDGMENTS

Thanks are given to Dicyt (USACH) and Fondecyt (Grant # 3980004) for financial support.

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